Embedding medium



Patented Dec. 23, 1941 UNITED, STATES PATENT OFFICE EMBEDDING MEDIUM Peter Gram-Pittsburgh, Pa., assignor to Stoner- Mudge, Inc., a. corporation of Pennsylvania.

No Drawing. "Application September 19, 1940, Serial No. 357,401

2 Claims. (01. '35-'20) mersing it in a suitable organic liquid such as ethyl alcohol or ,3 hydroxy-diethyl ether, known to the art as cellosolve. 'After a suitable period of immersion in such dehydrating liquid the specimen is removed and is at once immersed in a bath of a volatile liquid hydrocarbon, such as benzene, toluol, xylol, hi-flash naphtha, mineral spirits, etc. The hydrocarbon liquid containing the specimen is then poured into a suitable vessel containing melted paraffin wax, and the whole is then kept in an oven at a temperature not exceeding 150 F. until the volatile hydrocarbon has evaporated out of the resulting mixture and the specimen remains immersed in molten paraflin, free from air bubbles, trapped water, etc. The container of molten parafiin isthen removed from the oven and allowed to harden by cooling. Usually it is 'hardened by placing it in an ice bath. When completely set, the new solid cake of paraffin containing the specimen embedded within it is removed from the container and thin sections are shaved oflE by means of a microtome, down to that plane through the specimen which it is desired to investigate microscopically. These sections, either individually or in a continuous ribbon, as is well known, are removed to a glass microscope slide on which has been placed a layer of water containing small quantities of albumin, gelatin, or other organic protein deriva tive. If the sections are not flat it is customary to warm the solution to cause suflicient softening of the medium so that the sections lie down fiat.

or may be pressed fiat. The solution is then evaporated off at temperatures below the melting point of the embedding medium; the fiattened embedded sections adhere to the glassplate by virtue of the residual layer of albumin, gelatin, or other organic protein material. The embedding medium is then removed by dissolving in a solvent such as benzol, toluol, solvent naphtha, or other suitable hydrocarbon in order that the now waxand resin-free sections may subsedesired) before sealing under the usual thin glass cover slip in any of the resinous compositions such as Canada balsam or gum dammaniamiliarly employed for such mounting.

The paraflin wax used in the conventional procedure described above is when cold essentially crystalline and granular, as is well known, and

possesses little internal strength; so that, while reasonably thick sections have satisfactory strength to support and tightly hold the contained cross section of specimen, if the microtomed section be thinner than a value approximating 4-5 1. (micron), the internal strength of the paraflin will be insuflicient, and the sectioned sample will be likely to crumble and to be destroyed and rendered useless for microscopic examination.

The invention is found in modification of the parafiin wax by addition of other material to it,

in consequence of which the essentially crystalline nature of paraflin is changed, and the internal strength of the modified substance is increased to such extent that sections of 2-3y. thickness, or even less, may satisfactorily be prepared without crumbling or degradation.

; ,Another disadvantage attending the use of simple paraflin wax as the embedding medium (and it is onlywaxes alone that have hereto- 'fore been used for the purpose) is consequent upon the facts that its melting point is fairly low,

not exceeding 122 F., and that it does not have a sharply defined softening point. While'the mounted specimen may be cut satisfactorily by microtome at room temperatures, 'approximating 58-60" R, if the temperature approaches or exceeds '75-80 P. (which temperatures are hequently be differentially stained (if staining be 55 quently encountered during the greater part of the year), the simple paraflin medium becomes too.soft to be handled on the microtome; and tedious and cumbersome arrangements must then be provided to keep the specimen and the knife at a reduced temperature, low enough to insure satisfactory cutting. Extreme difficulty is often experienced in insuring these requisite lower temperatures during the entire process of cutting and mounting the specimen when embedded in simple parafiin wax. Accordingly, it

is another object of my invention so to modify the paraflin embedding medium that it shall re-'' the granular structure of the parafiinic embe'dding medium of the prior art, and to provide a medium of essentially homogeneous, amorphous texture.

My invention consists in the addition to simple paraffin wax of relatively small proportions, ranging from 2 to 40 per cent by weight, of a compatible resin, selected from a group consisting of the hydrogenated polymers of cyclopentadiene, di-cyclopentadiene, indene, and

cumarone, thus producing an embedding medium that, melting below 150 F., is firm and tough at ordinary temperatures, ranging upward to 95 F.; a medium that has an amorphous homogeneous texture; a medium that can be microtomed to a thickness less than 2-,3a and afford sections not too fragile to be handled satisfactorily and not susceptible to crumbling and deformation of the embedded specimen. Using my improved embedding medium, in the manner familiar to the art and essentially as previously described, it is possible to obtain sections of embedded biological specimens whose thickness is of a minuteness not heretofore obtainable, less than 3a, and to accomplish this without the necessity of laboriously cooling the embedded whole specimen, the microtome, and the knife.

The material that in the practice of my invention I add to paraflln is of a group that consists of the hydrogenated polymers of di-oleflnic hydroearbons and their oxygenated analogs, specifically cyclopentadiene. di-cyclopentadiene, cumarone, indene, and their ho'mologs and polymerizable alkyl and aryl derivatives. I have experimented widely, and have found no substance in this group that is not in some degree serviceable for my purposes; and I have every reason'to believe that all such substances areso serviceable, subject only to thelimitation that they be compatible with, and soluble in, paraflln and parafllnic hydrocarbons. Such polymers, interpolymers, and copolymers are well known to the chemical art, and are available on the market, as, for example, the substances bearing the trade names Nevillite (Nos. 1, 2, 3, and V), Piccolite, and hydogenated cumar.

For certain kinds of microtoming, I may also incorporate a few percent (usually 2, and not to exceed 5 per cent) of a natural or synthetic rubber or rubber-like polymeric substance, to impart slight elasticity and to eliminate even the last tendency to sharp fracturing of the paraflln. The synthetic rubber here contemplated is a material of a group that consists of polymerized chloroprene, polymerized butadiene, polymerized isobutylene, polymerized isoprene, polymerized divinyl acetylene, and their homologs.

I may substitute for a portion of the crystalline paraffin other paraflinic waxes, such as the branch-chained and naphthenic parafiins resulting from the purification and extraction of lubricating oils from petroleum.- Such petroleum waxes are to be had under the trade names of Barnsdall Wax, Petrowax, and Superla Wax.

Finally, I may add to my modified paraflln embedding medium a few per cent (not exceeding parts to 100 parts of the whole) of stearic acid, beeswax, spermacetti, camauba or other naturally occurring waxy substance, for conferring additional hardness and other desirable properties without loss of elasticity and mechanical strength.

The essential composition of my modified embedding medium will be made clear by the following illustrative examples. It will be understood that the proportions given are preferred proportions, and that the proportions may be varied within the over-all limits specified. The general composition I contemplate is as follows:

Substance Limits Percent 1 Hydrogenated polymer of cycle and di-cyclopentiene, indene, cumarone 2-40 2 Natural rubber or synthetic rubbery hydrocarbon polymer, such as polyisoprene, polychloroprene, polybutylene, polybutadiene, and their homologs" 05 3 Petrowax, Barnsdall Wax Superla Wax, and other branched chain or naphtlienic hydrocarbon waxes. 0-50 4 Btoario acid, beeswax, carnauba wax, spermacetti wax, gum dammar, and similar natural waxes and gums -0l0 5 Crystalline (straight chain) paraflln Balance Exemplary specific compositions are as follows:

Polymer of di-cyclopentadiene, hydrogenated--- 40 Example 5 Parts Paraffin 60 Petroleum wax 25 Polymerized di-eyclopentadiene, hydrogenated 15 Example 6 Parts Paraffi 50 Rubber 5 Superla w x 20 Beeswax 5 Spermacetti wax 5 Hydrogenated polymer of cyclopentadiene--- 15 Example 7 Parts Para 60 Rubber 2 Stearic acid 3 Gum dammar 10 Petroleum wax 15 Cumarone-lndene polymer, -hydrogenated 10 The preparation of my improved embedding medium offers no diillculty. I prefer to bring the paraflln into a condition of liquidity by application of heat, and, with or without mechanical agitation, to dissolve therein the resadded at the same time, or on subsequent melting of the mixture.

The term embedding medium is used throughout this specification in its technical sense, to designate specifically a material in which for purposes of mlcrotomy the specimen is initially embedded and which ultimately is dissolved away from the section when laid upon the microscope slide.

Having described my invention, and having pointed out the advantages attendant upon its use I claim as my invention:

1. An aromatic-hydrocarbon-soluble embedding medium for specimens for microscopic study susceptible to microtomy and removal by solution, consisting of paraffin wax modified by admixture with it of a substance of a group that consists of the hydrogenated polymers of cyclopen'tadiene, di-cyclopentadiene, cumarone, in-

dene,.. their homologs, and their polymerizable alkyl and aryl derivatives, together with a flex-Q ibilizing agent selected from a group that consists of rubber, polymerized chloroprene, poly-- merized butadlene, polymerized isobutylene, poly- 

